Chemokines, also known as “intercrines” and “SIS cytokines”, comprise a family of small secreted proteins (e.g., 70–100 amino acids and about 8–10 kiloDaltons) which attract and activate leukocytes and thereby aid in the stimulation and regulation of the immune system. The name “chemokine” is derived from chemotactic cytokine, and refers to the ability of these proteins to stimulate chemotaxis of leukocytes. Indeed, chemokines may comprise the main attractants for inflammatory cells into pathological tissues. See generally, Baggiolini et al., Annu. Rev. Immunol, 15: 675–705 (1997); and Baggiolini et al., Advances in Immunology, 55:97–179 (1994), both of which are incorporated by reference herein. While leukocytes comprise a rich source of chemokines, several chemokines are expressed in a multitude of tissues. Baggiolini et al. (1994), Table II.
Previously identified chemokines generally exhibit 20–70% amino acid identity to each other and contain four highly-conserved cysteine residues. Based on the relative position of the first two of these cysteine residues, chemokines have been further classified into two subfamilies. In the “C-X-C” or “α” subfamily, encoded by genes localized to human chromosome 4, the first two cysteines are separated by one amino acid. In the “C—C” or “β” subfamily, encoded by genes on human chromosome 17, the first two cysteines are adjacent. X-ray crystallography and NMR studies of several chemokines have indicated that, in each family, the first and third cysteines form a first disulfide bridge, and the second and fourth cysteines form a second disulfide bridge, strongly influencing the native conformation of the proteins. In humans alone, more than ten distinct sequences have been described for each chemokine subfamily. Chemokines of both subfamilies have characteristic leader sequences of twenty to twenty-five amino acids.
The C-X-C chemokines, which include IL-8, GROα/β/γ, platelet basic protein, Platelet Factor 4 (PF4), IP-10, NAP2, and others, share approximately 25% to 60% identity when any two amino acid sequences are compared (except for the GROα/β/γ members, which are 84–88% identical with each other). Most of the C-X-C chemokines (excluding IP-10 and Platelet Factor 4) share a common E-L-R tri-peptide motif upstream of the first two cysteine residues, and are potent stimulants of neutrophils, causing rapid shape change, chemotaxis, respiratory bursts, and degranulation. These effects are mediated by seven-transmembrane-domain rhodopsin-like G protein-coupled receptors; a receptor specific for IL-8 has been cloned by Holmes et al., Science, 253:1278–80 (1991), while a similar receptor (77% identity) which recognizes IL-8, GRO and NAP2 has been cloned by Murphy and Tiffany, Science, 253:1280–83 (1991). Progressive truncation of the N-terminal amino acid sequence of certain C-X-C chemokines, including IL-8, is associated with marked increases in activity.
The C—C chemokines, which include Macrophage Inflammatory Proteins MIP-1α and MIP-1β, Monocyte chemoattractant proteins 1, 2, 3, and 4 (MCP-1/2/3/4), RANTES, I-309, eotaxin, TARC, and others, share 25% to 70% amino acid identity with each other. Previously-identified C—C chemokines activate monocytes, causing calcium flux and chemotaxis. More selective effects are seen on lymphocytes, for example, T lymphocytes, which respond best to RANTES. Several seven-transmembrane-domain G protein-coupled receptors for C—C chemokines have been cloned to date, including a C—C chemokine receptor-1 (CCR1) which recognizes, e.g., MIP-1α and RANTES (Neote et al., Cell, 72:415–425 (1993)); a CCR2 receptor which has two splice variants and which recognizes, e.g., MCP-1 (Charo et al., Proc. Nat. Acad. Sci., 91:2752–56 (1994)); CCR3, which recognizes, e.g., eotaxin, RANTES, and MCP-3 (Combadiere, J. Biol. Chem., 270:16491 (1995)); CCR4, which recognizes MIP-1α, RANTES, and MCP-1 (Power et al., J. Biol. Chem., 270:19495 (1995)); and CCR5, which recognizes MIP-1α, MIP-1β, and RANTES (Samson et al., Biochemstry, 35:3362 (1996)). Several CC chemokines have been shown to act as attractants for activated T lymphocytes. See Baggiolini et al. (1997).
Truncation of the N-terminal amino acid sequence of certain C—C chemokines also has been associated with alterations in activity. For example, mature RANTES (1–68) is processed by CD26 (a dipeptidyl aminopeptidase specific for the sequence NH2-X-Pro- . . . ) to generate a RANTES (3–68) form that is capable of interacting with and transducing a signal through CCR5 (like the RANTES (1–68) form), but is one hundred-fold reduced in its capacity to stimulate through the receptor CCR1. See Proost et al., J. Biol. Chem., 273(13): 7222–7227 (1998); and Oravecz et al., J. Exp. Med., 186: 1865–1872 (1997). U.S. Pat. Nos. 5,459,128, 5,705,360, and 5,739,103 to Rollins and Zhang purport to describe N-terminal deletions of chemokine MCP-1 that inhibit receptor binding to the corresponding endogenous chemokine.
The roles of a number of chemokines, particularly IL-8, have been well documented in various pathological conditions. See generally Baggiolini et al. (1994), supra, Table VII. Psoriasis, for example, has been linked to over-production of IL-8, and several studies have observed high levels of IL-8 in the synovial fluid of inflamed joints of patients suffering from rheumatic diseases, osteoarthritis, and gout.
The role of C—C chemokines in pathological conditions also has been documented, albeit less comprehensively than the role of IL-8. For example, the concentration of MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases. The MCP-1 dependent influx of mononuclear phagocytes may be an important event in the development of idiopathic pulmonary fibrosis. The role of C—C chemokines in the recruitment of monocytes into atherosclerotic areas is currently of intense interest, with enhanced MCP-1 expression having been detected in macrophage-rich arterial wall areas but not in normal arterial tissue. Expression of MCP-1 in malignant cells has been shown to suppress the ability of such cells to form tumors in vivo. (See U.S. Pat. No. 5,179,078, incorporated herein by reference.) A need therefore exists for the identification and characterization of additional C—C chemokines, to further elucidate the role of this important family of molecules in pathological conditions, and to develop improved treatments for such conditions utilizing chemokine-derived products.
With respect to the involvement of chemokines in allergic diseases, interest has focused on chemokines belonging to the CC family, such as RANTES, eotaxin, eotaxin-2, MCP-3 and MCP4, because of their ability to cause migration of human eosinophils in vitro and in vivo.
The ability of these chemokines to selectively activate human eosinophil migration appears to be due primarily to their activation of chemokine receptor CCR3. A need exists to elucidate the involvement of these and other chemokines in eosinophil stimulation and activation, to facilitate better treatments for late-phase allergic reactions, such as asthma [see Aalbers et al., Eur. Respir. J., 6:840(1993); and Frigas et al., J. Allergy Clin. Immunol., 77:527(1986)], in which eosinophil activation and migration have been implicated.
Chemokines of the C—C subfamily have been shown to possess utility in medical imaging, e.g., for imaging sites of infection, inflammation, and other sites having C—C chemokine receptor molecules. See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778, incorporated herein by reference. Such methods involve chemical attachment of a labeling agent (e.g., a radioactive isotope) to the C—C chemokine using art recognized techniques (see, e.g., U.S. Pat. Nos. 4,965,392 and 5,037,630, incorporated herein by reference), administration of the labeled chemokine to a subject in a pharmaceutically acceptable carrier, allowing the labeled chemokine to accumulate at a target site, and imaging the labeled chemokine in vivo at the target site. A need in the art exists for additional new C—C chemokines to increase the available arsenal of medical imaging tools.
The C—C chemokines RANTES, MIP-α, and MIP-1μ also have been shown to be the primary mediators of the suppressive effect of human T cells on the human immunodeficiency virus (HIV), the agent responsible for causing human Acquired Immune Deficiency Syndrome (AIDS). These chemokines show a dose-dependent ability to inhibit specific strains of HIV from infecting cultured T cell lines [Cocchi et al., Science, 270:1811 (1995)]. In addition, International patent publication number WO 97/44462, filed by Institut Pasteur, describes the use of fragments and analogs of the chemokine RANTES as antagonists, to block RANTES interaction with its receptors, for the purpose of suppressing HIV. The C-X-C chemokine stromal derived factor-1 (SDF-1) also is capable of blocking infection by T-tropic HIV-1 strains. See Winkler et al., Science, 279:389–393 (1998). However, the processes through which chemokines exert their protective effects have not been fully elucidated, and these chemokines in fact may stimulate HIV replication in cells exposed to the chemokines before HIV infection. See Kelly et al., J. Immunol., 160:3091–3095 (1998). Moreover, not all tested strains of the virus are equally susceptible to the inhibitory effects of chemokines; therefore, a need exists for additional C—C chemokines for use as inhibitors of strains of HIV.
Similarly, it has been established that certain chemokine receptors such as CCR5 [International Patent Publication No. WO 97/44055, published 27 Nov. 1997], CCR8, CCR2, and CXCR4) are essential co-receptors (with the CD4 receptor) for mV-1 entry into susceptible cells, and that progression to AIDS is delayed in patients having certain variant alleles of these receptors. A need exists for additional therapeutics to inhibit HIV-1 infection and/or proliferation by interfering with HIV-1 entry and/or proliferation in susceptible cells.
More generally, due to the importance of chemokines as mediators of chemotaxis and inflammation, a need exists for the identification and isolation of new members of the chemokine family to facilitate modulation of inflammatory and immune responses.
For example, substances that promote inflammation may promote the healing of wounds or the speed of recovery from conditions such as pneumonia, where inflammation is important to eradication of infection. Modulation of inflammation is similarly important in pathological conditions manifested by inflammation. Crohn's disease, manifested by chronic inflammation of all layers of the bowel, pain, and diarrhea, is one such pathological condition. The failure rate of drug therapy for Crohn's disease is relatively high, and the disease is often recurrent even in patients receiving surgical intervention. The identification, isolation, and characterization of novel chemokines facilitates modulation of inflammation.
Similarly, substances that induce an immune response may promote palliation or healing of any number of pathological conditions. Due to the important role of leukocytes (e.g., neutrophils and monocytes) in cell-mediated immune responses, and due to the established role of chemokines in leukocyte chemotaxis, a need exists for the identification and isolation of new chemokines to facilitate modulation of immune responses.
Additionally, the established correlation between chemokine expression and inflammatory conditions and disease states provides diagnostic and prognostic indications for the use of chemokines, as well as for antibody substances that are specifically immunoreactive with chemokines; a need exists for the identification and isolation of new chemokines to facilitate such diagnostic and prognostic indications.
In addition to their ability to attract and activate leukocytes, some chemokines, such as L-8, have been shown to be capable of affecting the proliferation of non-leukocytic cells. See Tuschil, J. Invest. Dermatol., 99:294–298 (1992). A need exists for the identification and isolation of new chemokines to facilitate modulation of such cell proliferation.
It will also be apparent from the foregoing discussion of chemokine activities that a need exists for modulators of chemokine activities, to inhibit the effects of endogenously-produced chemokines and/or to promote the activities of endogenously-produced or exogenously administered chemokines. Such modulators typically include small molecules, peptides, chemokine fragments and analogs, and/or antibody substances. Chemokine inhibitors interfere with chemokine signal transduction, i.e., by binding chemokine molecules, by competitively or non-competitively binding chemokine receptors, and/or by interfering with signal transduction downstream from the chemokine receptors. A need exists in the art for effective assays to rapidly screen putative chemokine modulators for modulating activity.
For all of the aforementioned reasons, a need exists for recombinant methods of production of newly discovered chemokines, which methods facilitate clinical applications involving the chemokines and chemokine inhibitors.